1. Field of the Invention
The present invention relates to a new process for effecting the acylation step in amide formation, especially during peptide synthesis. More specifically, the invention relates to the use of a compound having the formula ##STR2## and N-oxides thereof and salts thereof wherein R.sub.1 and R.sub.2 taken together with the carbon atoms to which they are attached form a heteroaryl ring wherein said heteroaryl ring is an oxygen, sulfur or nitrogen containing heteroaromatic containing from 3 and up to a total of 13 ring carbon atoms, said heteroaryl may be unsubstituted or substituted with lower alkyl or an electron-donating group;
Y is O, NR.sub.4, CR.sub.4 R.sub.5 ; PA1 R.sub.5 is independently hydrogen or lower alkyl; PA1 X is CR.sub.6 R.sub.7 or NR.sub.6 ; PA1 R.sub.6 or R.sub.7 are independently hydrogen or lower alkyl; or R.sub.6 and R.sub.7 taken together form an oxo group or when n=O, R.sub.4 and R.sub.6 taken together may form a bond between the nitrogen or carbon atom of Y and the nitrogen or carbon atom of X; PA1 Q is (CR.sub.8 R.sub.9) or (NR.sub.8); PA1 when n is 1, R.sub.4 and R.sub.8 taken together may form a bond between the ring carbon or nitrogen atom of Q and the ring carbon or nitrogen atom of R.sub.8 ; PA1 n is 0, 1 or 2; PA1 R.sub.3 is hydrogen, lower alkyl carbonyl, aryl carbonyl, lower aryl alkyl carbonyl, ##STR3## a positively charged electron withdrawing group, SO.sub.2 R.sub.14, or ##STR4## R.sub.14 is lower alkyl, aryl or lower arylalkyl; q is 0-3; R.sub.8 and R.sub.9 are independently hydrogen or lower alkyl or R.sub.7 and R.sub.8 taken together with the carbon to which they are attached form an aryl ring, AA.sub.1 is an amino acid and BLK is an amino protecting group, and m is 0 or 1. PA1 R.sub.9 is hydrogen or lower alkyl and R.sub.1, R.sub.2, X, OR.sub.3 are as defined hereinabove. Examples of the above formula include: ##STR13## PA1 A is N or CR.sub.15 ; PA1 D is CR.sub.16 or N; PA1 E is CR.sub.17 or N; PA1 G is CR.sub.18 or N; and PA1 R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are independently hydrogen or lower alkyl or an electron donating group or R.sub.16 and R.sub.17 taken together form an aryl ring, but at least one of A, D, E, G is N. PA1 R.sub.15 is Me, Et, i-Pr, iPr.sub.2 N, or CMe.sub.3 PA1 J is O, or S(O)p, and PA1 p is 0, 1 or 2. PA1 1) a base labile N.alpha.-amino acid protecting group such as FMOC, and the like. PA1 2) protecting groups removed by acid, such as Boc, TEOC, Aoc, Adoc, Mcb, Bpoc, Azoc, Ddz, Poc, Cbz, 2-furanmethyloxycarbonyl (Foc), p-methoxybenzyloxycarbonyl (Moz), Nps, and the like. PA1 3) protecting groups removed by hydrogenation such as Dts, Cbz. PA1 4) protecting groups removed by nucleophiles, such as Bspoc, Bsmoc and Nps and the like. PA1 5) protecting groups derived from carboxylic acids, such as formyl, acetyl, trifluoroacetyl and the like, which are removed by acid, base or nucleophiles. PA1 BLK is an amino protecting group PA1 AA is an amino acid and PA1 M is halo or ##STR27## wherein R.sub.20 is independently halo, lower alkyl, nitro, cyano or other electron withdrawing groups and n is 0-5. When n is 0, the phenoxy ester is unsubstituted. PA1 1) protection of the free carboxyl group in a first amino acid or a first peptide, unless the amino acid or peptide is anchored to a solid support. PA1 2) protection of the free amino group of a second amino acid or peptide. PA1 3) protection of the side chains, if necessary. PA1 4) coupling the first amino acid or peptide with the second amino acid or peptide in the presence of compounds of Formula I. PA1 5) removal of the protecting groups.
The present invention also relates to novel compounds encompassed by the above-identified formula.
2. Description of the Prior Art
Polypeptides are useful as medicaments. In recent years, peptides have been found to be useful in combatting various diseases, such as cancer, diabetes, plant toxins and the like. Additionally, peptides have shown specific activity as growth promoters, suppressants, antibiotics, insecticides, contraceptives, anti-hypertensives, sleep-inducers, anti-depressants, analgesics, etc. The list is long and varied.
As more and more polypeptides become of medicinal importance, there is an increasing incentive to improve the methods by which they may be synthesized. Currently, syntheses of peptides are in solution by classical or various repetitive methods. Alternatively, peptides may be prepared on a solid support (Merrifield method). These are all popular techniques in synthesizing peptides from the coupling of two or more amino acids, in synthesizing larger peptides from the coupling of amino acids with smaller peptides or in the coupling of smaller peptides. Solution methods have the advantage of being easily monitored, allowing purification of intermediates, if necessary, at any stage. A major drawback, however, is the relative slow pace of synthesis, with each step being carried out manually.
The major advantage of the Merrifield method is its easy automation so that unattended, computer-controlled machine synthesis is possible. Unfortunately, the method suffers from an inherent deficiency due to the insoluble nature of the support on which the synthesis proceeds. Unless each acylation step occurs with approximately 100% efficiency, mixtures will inevitably be built up on the polymer. The longer the chain, the greater will be the contamination by undesired side reactions. Side products produced in such reactions remain to contaminate the desired product when it is removed from the polymeric matrix at the end of the cycle. These current techniques are not useful in preparing peptides of greater than 20-30 residues; separation of side products from the desired product becomes increasingly difficult when larger peptides are synthesized.
For very long segments (50 or more amino acids), therefore, current methods are not satisfactory. Often, mixtures are obtained of such forbidding complexity that it may be difficult or impossible to isolate the desired peptide.
The problems enumerated hereinabove may be eliminated if the proper derivatives of the underlying amino acids and/or the proper conditions for the coupling reaction could be found. Protecting groups, such as t-butyloxy-carbonyl (t-Boc) or N-.alpha.-(9-fluorenylmethyl)oxycarbonyl (Fmoc), have been used to minimize side reactions. But, additionally, other aspects of the coupling reaction must also be taken into consideration, such as the peptide coupling additive to be used in the coupling reaction.
Additives generally inhibit side reactions and reduce racemization. Heretofore, the most common peptide coupling additive used during peptide coupling for both solutions and solid phase syntheses is 1-hydroxybenzotriazole (HOBt). This reagent has been used either in combination with a carbodimide or other coupling agent or built into a stand-alone reagent, such as 1-benzotriazolyoxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or an analogous uronium salt. HOBt is applicable to both stepwise and segment condensations. However, many cases have been encountered in which HOBt is ineffective, possibly because of steric effects, or low basicity of the amino component. Especially problematic are segment couplings at amino acid units other than glycine or proline, since the problem of racemization may be severe. The related N-hydroxybenzotriazinone (HOOBt) may provide better protection against racemization, but it is rarely used due to competing side reactions involving ring openings.
However, the present inventor has discovered that compounds of Formula I are effective as peptide coupling additives in both stepwise (batch and continuous flow) and segment condensations to peptide syntheses. Compounds of Formula I overcame deficiencies of the additives used heretofore. Compounds of the present invention, as a peptide coupling additive, have the ability to accelerate the reaction, or provide cleaner processes, higher yields and less racemization. The products formed with the use of compounds of the present invention tend to be purer than those made by methods used heretofore. Yet, the reaction conditions are very mild, and the reagents used are commercially available and/or easy to prepare.
Furthermore, compounds within the scope of the present invention have an additional benefit and provide a visual indication of the reaction endpoint. For example, HOAt or 1-hydroxy-7-azabenzotriazole in the presence of an amino acid or peptide ester, is converted to its anion, which is colored. As coupling proceeds, the color fades and then disappears completely when the coupling reaction is completed. By watching for the disappearance of the color, the researcher knows when the coupling reaction is completed. The researcher does not need to monitor the reaction to determine when the coupling reaction is completed. He does not need to wait an unspecified amount of time for the reaction to be completed. Thus, by using the compound of the present invention the researcher, as a result, can use his time more efficiently.